CD9 co-operation with syndecan-1 is required for a major staphylococcal adhesion pathway.

Epithelial colonization is a critical first step in bacterial pathogenesis. Staphylococcus aureus can utilize several host factors to associate with cells, including α5ß1 integrin and heparan sulfate proteoglycans, such as the syndecans. Here, we demonstrate that a partner protein of both integrins and syndecans, the host membrane adapter protein tetraspanin CD9, is essential for syndecan-mediated staphylococcal adhesion. Fibronectin is also essential in this process, while integrins are only critical for post-adhesion entry into human epithelial cells. Treatment of epithelial cells with CD9-derived peptide or heparin caused significant reductions in staphylococcal adherence, dependent on both CD9 and syndecan-1. Exogenous fibronectin caused a CD9-dependent increase in staphylococcal adhesion, whereas blockade of ß1 integrins did not affect adhesion but did reduce the subsequent internalization of adhered bacteria. CD9 disruption or deletion increased ß1 integrin-mediated internalization, suggesting that CD9 coordinates sequential staphylococcal adhesion and internalization. CD9 controls staphylococcal adhesion through syndecan-1, using a mechanism that likely requires CD9-mediated syndecan organization to correctly display fibronectin at the host cell surface. We propose that CD9-derived peptides or heparin analogs could be developed as anti-adhesion treatments to inhibit the initial stages of staphylococcal pathogenesis. IMPORTANCE Staphylococcus aureus infection is a significant cause of disease and morbidity. Staphylococci utilize multiple adhesion pathways to associate with epithelial cells, including interactions with proteoglycans or ß1 integrins through a fibronectin bridge. Interference with another host protein, tetraspanin CD9, halves staphylococcal adherence to epithelial cells, although CD9 does not interact directly with bacteria. Here, we define the role of CD9 in staphylococcal adherence and uptake, observing that CD9 coordinates syndecan-1, fibronectin, and ß1 integrins to allow efficient staphylococcal infection. Two treatments that disrupt this action are effective and may provide an alternative to antibiotics. We provide insights into the mechanisms that underlie staphylococcal infection of host cells, linking two known adhesion pathways together through CD9 for the first time.

Tetraspanin CD9-derived peptides inhibit Pseudomonas aeruginosa corneal infection and aid in wound healing of corneal epithelial cells.

Pseudomonas aeruginosa is a leading cause of corneal infection both within India and globally, often causing a loss of vision. Increasing antimicrobial resistance among the bacteria is making its treatment more difficult. Preventing initial bacterial adherence to the host membrane has been explored here to reduce infection of the cornea. Synthetic peptides derived from human tetraspanin CD9 have been shown to reduce infection in corneal cells both in vitro, ex vivo and in vivo. We found constitutive expression of CD9 in immortalized human corneal epithelial cells by flow cytometry and immunocytochemistry. The synthetic peptides derived from CD9 significantly reduced bacterial adherence to cultured corneal epithelial cells and ex vivo human cadaveric corneas as determined by colony forming units. The peptides also significantly reduced bacterial burden in a murine model of Pseudomonas keratitis and lowered the cellular infiltration in the corneal stroma. Additionally, the peptides aided corneal wound healing in uninfected C57BL/6 mice compared to control mice. These potential therapeutics had no effect on cell viability or proliferation of corneal epithelial cells and have the potential to be developed as an alternative therapeutic intervention.

Tetraspanin Cd9b plays a role in fertility in zebrafish.

In mice, CD9 expression on the egg is required for efficient sperm-egg fusion and no effects on ovulation or male fertility are observed in CD9 null animals. Here we show that cd9b knockout zebrafish also appear to have fertility defects. In contrast to mice, fewer eggs were laid by cd9b knockout zebrafish pairs and, of the eggs laid, a lower percentage were fertilised. These effects could not be linked to primordial germ cell numbers or migration as these were not altered in the cd9b mutants. The decrease in egg numbers could be rescued by exchanging either cd9b knockout partner, male or female, for a wildtype partner. However, the fertilisation defect was only rescued by crossing a cd9b knockout female with a wildtype male. To exclude effects of mating behaviour we analysed clutch size and fertilisation using in vitro fertilisation techniques. Number of eggs and fertilisation rates were significantly reduced in the cd9b mutants suggesting the fertility defects are not solely due to courtship behaviours. Our results indicate that CD9 plays a more complex role in fish fertility than in mammals, with effects in both males and females.

Tetraspanin Cd9b and Cxcl12a/Cxcr4b have a synergistic effect on the control of collective cell migration.

Collective cell migration is essential for embryonic development and homeostatic processes. During zebrafish development, the posterior lateral line primordium (pLLP) navigates along the embryo flank by collective cell migration. The chemokine receptors, Cxcr4b and Cxcr7b, as well as their cognate ligand, Cxcl12a, are essential for this process. We corroborate that knockdown of the zebrafish cd9 tetraspanin orthologue, cd9b, results in mild pLL abnormalities. Through generation of CRISPR and TALEN mutants, we show that cd9a and cd9b function partially redundantly in pLLP migration, which is delayed in the cd9b single and cd9a; cd9b double mutants. This delay led to a transient reduction in neuromast numbers. Loss of both Cd9a and Cd9b sensitized embryos to reduced Cxcr4b and Cxcl12a levels. Together these results provide evidence that Cd9 modulates collective cell migration of the pLLP during zebrafish development. One interpretation of these observations is that Cd9 contributes to more effective chemokine signalling.

ACE2-Independent Interaction of SARS-CoV-2 Spike Protein with Human Epithelial Cells Is Inhibited by Unfractionated Heparin.

Coronaviruses such as SARS-CoV-2, which is responsible for COVID-19, depend on virus spike protein binding to host cell receptors to cause infection. The SARS-CoV-2 spike protein binds primarily to ACE2 on target cells and is then processed by membrane proteases, including TMPRSS2, leading to viral internalisation or fusion with the plasma membrane. It has been suggested, however, that receptors other than ACE2 may be involved in virus binding. We have investigated the interactions of recombinant versions of the spike protein with human epithelial cell lines that express low/very low levels of ACE2 and TMPRSS2 in a proxy assay for interaction with host cells. A tagged form of the spike protein containing the S1 and S2 regions bound in a temperature-dependent manner to all cell lines, whereas the S1 region alone and the receptor-binding domain (RBD) interacted only weakly. Spike protein associated with cells independently of ACE2 and TMPRSS2, while RBD required the presence of high levels of ACE2 for interaction. As the spike protein has previously been shown to bind heparin, a soluble glycosaminoglycan, we tested the effects of various heparins on ACE2-independent spike protein interaction with cells. Unfractionated heparin inhibited spike protein interaction with an IC50 value of <0.05 U/mL, whereas two low-molecular-weight heparins were less effective. A mutant form of the spike protein, lacking the arginine-rich putative furin cleavage site, interacted only weakly with cells and had a lower affinity for unfractionated and low-molecular-weight heparin than the wild-type spike protein. This suggests that the furin cleavage site might also be a heparin-binding site and potentially important for interactions with host cells. The glycosaminoglycans heparan sulphate and dermatan sulphate, but not chondroitin sulphate, also inhibited the binding of spike protein, indicating that it might bind to one or both of these glycosaminoglycans on the surface of target cells.

Matriptase activation of Gq drives epithelial disruption and inflammation via RSK and DUOX.

Epithelial tissues are primed to respond to insults by activating epithelial cell motility and rapid inflammation. Such responses are also elicited upon overexpression of the membrane-bound protease, Matriptase, or mutation of its inhibitor, Hai1. Unrestricted Matriptase activity also predisposes to carcinoma. How Matriptase leads to these cellular outcomes is unknown. We demonstrate that zebrafish hai1a mutants show increased H2O2, NfκB signalling, and IP3R -mediated calcium flashes, and that these promote inflammation, but do not generate epithelial cell motility. In contrast, inhibition of the Gq subunit in hai1a mutants rescues both the inflammation and epithelial phenotypes, with the latter recapitulated by the DAG analogue, PMA. We demonstrate that hai1a has elevated MAPK pathway activity, inhibition of which rescues the epidermal defects. Finally, we identify RSK kinases as MAPK targets disrupting adherens junctions in hai1a mutants. Our work maps novel signalling cascades mediating the potent effects of Matriptase on epithelia, with implications for tissue damage response and carcinoma progression.

Cancer occurs when normal processes in the cell become corrupted or unregulated. Many proteins can contribute, including one enzyme called Matriptase that cuts other proteins at specific sites. Matriptase activity is tightly controlled by a protein called Hai1. In mice and zebrafish, when Hai1 cannot adequately control Matriptase activity, invasive cancers with severe inflammation develop. However, it is unclear how unregulated Matriptase leads to both inflammation and cancer invasion. One outcome of Matriptase activity is removal of proteins called Cadherins from the cell surface. These proteins have a role in cell adhesion: they act like glue to stick cells together. Without them, cells can dissociate from a tissue and move away, a critical step in cancer cells invading other organs. However, it is unknown exactly how Matriptase triggers the removal of Cadherins from the cell surface to promote invasion. Previous work has shown that Matriptase switches on a receptor called Proteinase-activated receptor 2, or Par2 for short, which is known to activate many enzymes, including one called phospholipase C. When activated, this enzyme releases two signals into the cell: a sugar called inositol triphosphate, IP3; and a lipid or fat called diacylglycerol, DAG. It is possible that these two signals have a role to play in how Matriptase removes Cadherins from the cell surface. To find out, Ma et al. mapped the effects of Matriptase in zebrafish lacking the Hai1 protein. This revealed that Matriptase increases IP3 and DAG levels, which initiate both inflammation and invasion. IP3 promotes inflammation by switching on pro-inflammatory signals inside the cell such as the chemical hydrogen peroxide. At the same time, DAG promotes cell invasion by activating a well-known cancer signalling pathway called MAPK. This pathway activates a protein called RSK. Ma et al. show that this protein is required to remove Cadherins from the surface of cells, thus connecting Matriptase's activation of phospholipase C with its role in disrupting cell adhesion. An increase in the ratio of Matriptase to HAI-1 (the human equivalent of Hai1) is present in many cancers. For this reason, the signal cascades described by Ma et al. may be of interest in developing treatments for these cancers. Understanding how these signals work together could lead to more direct targeted anti-cancer approaches in the future.

Corneal Infection Models: Tools to Investigate the Role of Biofilms in Bacterial Keratitis.

Bacterial keratitis is a corneal infection which may cause visual impairment or even loss of the infected eye. It remains a major cause of blindness in the developing world. Staphylococcus aureus and Pseudomonas aeruginosa are common causative agents and these bacterial species are known to colonise the corneal surface as biofilm populations. Biofilms are complex bacterial communities encased in an extracellular polymeric matrix and are notoriously difficult to eradicate once established. Biofilm bacteria exhibit different phenotypic characteristics from their planktonic counterparts, including an increased resistance to antibiotics and the host immune response. Therefore, understanding the role of biofilms will be essential in the development of new ophthalmic antimicrobials. A brief overview of biofilm-specific resistance mechanisms is provided, but this is a highly multifactorial and rapidly expanding field that warrants further research. Progression in this field is dependent on the development of suitable biofilm models that acknowledge the complexity of the ocular environment. Abiotic models of biofilm formation (where biofilms are studied on non-living surfaces) currently dominate the literature, but co-culture infection models are beginning to emerge. In vitro, ex vivo and in vivo corneal infection models have now been reported which use a variety of different experimental techniques and animal models. In this review, we will discuss existing corneal infection models and their application in the study of biofilms and host-pathogen interactions at the corneal surface.

Tetraspanins are involved in Burkholderia pseudomallei-induced cell-to-cell fusion of phagocytic and non-phagocytic cells.

Tetraspanins are four-span transmembrane proteins of host cells that facilitate infections by many pathogens. Burkholderia pseudomallei is an intracellular bacterium and the causative agent of melioidosis, a severe disease in tropical regions. This study investigated the role of tetraspanins in B. pseudomallei infection. We used flow cytometry to determine tetraspanins CD9, CD63, and CD81 expression on A549 and J774A.1 cells. Their roles in B. pseudomallei infection were investigated in vitro using monoclonal antibodies (MAbs) and recombinant large extracellular loop (EC2) proteins to pretreat cells before infection. Knockout of CD9 and CD81 in cells was performed using CRISPR Cas9 to confirm the role of tetraspanins. Pretreatment of A549 cells with MAb against CD9 and CD9-EC2 significantly enhanced B. pseudomallei internalization, but MAb against CD81 and CD81-EC2 inhibited MNGC formation. Reduction of MNGC formation was consistently observed in J774.A1 cells pretreated with MAbs specific to CD9 and CD81 and with CD9-EC2 and CD81-EC2. Data from knockout experiments confirmed that CD9 enhanced bacterial internalization and that CD81 inhibited MNGC formation. Our data indicate that tetraspanins are host cellular factors that mediated internalization and membrane fusion during B. pseudomallei infection. Tetraspanins may be the potential therapeutic targets for melioidosis.

Correction to: A role for tetraspanin proteins in regulating fusion induced by Burkholderia thailandensis.

In the original article, incorrect  figures were published with incorrect captions. The correct figures and captions are given below.

A role for tetraspanin proteins in regulating fusion induced by Burkholderia thailandensis.

Burkholderia pseudomallei is the causative agent of melioidosis, a disease with high morbidity that is endemic in South East Asia and northern Australia. An unusual feature of the bacterium is its ability to induce multinucleated giant cell formation (MNGC), which appears to be related to bacterial pathogenicity. The mechanism of MNGC formation is not fully understood, but host cell factors as well as known bacterial virulence determinants are likely to contribute. Since members of the tetraspanin family of membrane proteins are involved in various types of cell:cell fusion, their role in MNGC formation induced by Burkholderia thailandensis, a mildly pathogenic species closely related to B. pseudomallei, was investigated. The effect of antibodies to tetraspanins CD9, CD81, and CD63 in MNGC formation induced by B. thailandensis in infected mouse J774.2 and RAW macrophage cell lines was assessed along with that of recombinant proteins corresponding to the large extracellular domain (EC2) of the tetraspanins. B. thailandensis-induced fusion was also examined in macrophages derived from CD9 null and corresponding WT mice, and in J774.2 macrophages over-expressing CD9. Antibodies to CD9 and CD81 promoted MNGC formation induced by B. thailandensis, whereas EC2 proteins of CD9, CD81, and CD63 inhibited MNGC formation. Enhanced MNGC formation was observed in CD9 null macrophages, whereas a decrease in MNGC formation was associated with overexpression of CD9. Overall our findings show that tetraspanins are involved in MNGC formation induced by B. thailandensis and by implication, B. pseudomallei, with CD9 and CD81 acting as negative regulators of this process.

Monocyte Subsets Have Distinct Patterns of Tetraspanin Expression and Different Capacities to Form Multinucleate Giant Cells.

Monocytes are able to undergo homotypic fusion to produce different types of multinucleated giant cells, such as Langhans giant cells in response to M. tuberculosis infection or foreign body giant cells in response to implanted biomaterials. Monocyte fusion is highly coordinated and complex, with various soluble, intracellular, and cell-surface components mediating different stages of the process. Tetraspanins, such as CD9, CD63, and CD81, are known to be involved in cell:cell fusion and have been suggested to play a role in regulating homotypic monocyte fusion. However, peripheral human monocytes are not homogenous: they exist as a heterogeneous population consisting of three subsets, classical (CD14++CD16-), intermediate (CD14++CD16+), and non-classical (CD14+CD16+), at steady state. During infection with mycobacteria, the circulating populations of intermediate and non-classical monocytes increase, suggesting they may play a role in the disease outcome. Human monocytes were separated into subsets and then induced to fuse using concanavalin A. The intermediate monocytes were able to fuse faster and form significantly larger giant cells than the other subsets. When antibodies targeting tetraspanins were added, the intermediate monocytes responded to anti-CD63 by forming smaller giant cells, suggesting an involvement of tetraspanins in fusion for at least this subset. However, the expression of fusion-associated tetraspanins on monocyte subsets did not correlate with the extent of fusion or with the inhibition by tetraspanin antibody. We also identified a CD9High and a CD9Low monocyte population within the classical subset. The CD9High classical monocytes expressed higher levels of tetraspanin CD151 compared to CD9Low classical monocytes but the CD9High classical subset did not exhibit greater potential to fuse and the role of these cells in immunity remains unknown. With the exception of dendrocyte-expressed seven transmembrane protein, which was expressed at higher levels on the intermediate monocyte subset, the expression of fusion-related proteins between the subsets did not clearly correlate with their ability to fuse. We also did not observe any clear correlation between giant cell formation and the expression of pro-inflammatory or fusogenic cytokines. Although tetraspanin expression appears to be important for the fusion of intermediate monocytes, the control of multinucleate giant cell formation remains obscure.

The Use of Plant-Derived Ribosome Inactivating Proteins in Immunotoxin Development: Past, Present and Future Generations.

Ribosome inactivating proteins (RIPs) form a class of toxins that was identified over a century ago. They continue to fascinate scientists and the public due to their very high activity and long-term stability which might find useful applications in the therapeutic killing of unwanted cells but can also be used in acts of terror. We will focus our review on the canonical plant-derived RIPs which display ribosomal RNA N-glycosidase activity and irreversibly inhibit protein synthesis by cleaving the 28S ribosomal RNA of the large 60S subunit of eukaryotic ribosomes. We will place particular emphasis on therapeutic applications and the generation of immunotoxins by coupling antibodies to RIPs in an attempt to target specific cells. Several generations of immunotoxins have been developed and we will review their optimisation as well as their use and limitations in pre-clinical and clinical trials. Finally, we endeavour to provide a perspective on potential future developments for the therapeutic use of immunotoxins.

A role for the tetraspanin proteins in Salmonella infection of human macrophages.

OBJECTIVE: Infected macrophages play a role in the dissemination of Salmonella and may serve as a reservoir of infection in asymptomatic carriers. However, relatively little is known about the early molecular interactions of the bacteria with these cells. We have recently shown that members of the tetraspanin family of membrane proteins are involved in the initial adhesion of a range of bacteria to host cells. This study investigated the role of tetraspanins in Salmonella enterica subsp. enterica serovar Typhimurium (S. Typhimurium) infection of human monocyte-derived macrophages (MDM). METHODS: The role of tetraspanins was studied by the use of tetraspanins recombinant proteins as well as monoclonal antibodies targeted against different tetraspanins. Knockdown of the tetraspanin CD63 was carried out by siRNA to further study the role of CD63 in Salmonella uptake. RESULTS: Recombinant proteins representing the large extracellular domains of tetraspanins inhibited binding of S. Typhimurium to human MDM by ∼50%, whereas tetraspanin-specific antibodies showed varying effects, with some enhancing (anti-CD37) and some inhibiting (anti-CD81, anti-CD63) binding. Inhibition of the S. Typhimurium-MDM interaction by anti-CD63 mAb appeared to be mediated by antibody induced internalization, suggesting that surface expression of CD63 is required for S. Typhimurium binding. Knockdown of CD63 in human MDM using siRNA greatly reduced S. Typhimurium binding, confirming the importance of CD63. However, ectopic expression of CD63 in the non-phagocytic cell line HEK293 was insufficient to mediate bacterial binding. CONCLUSION: Bacterial adhesion is the first step in infection by pathogens that invade and replicate within host cells. Taken together, the results here describe a role for tetraspanins in binding of S. Typhimurium to human macrophages and highlight the particular importance of CD63 in this process.

Tspan2: a tetraspanin protein involved in oligodendrogenesis and cancer metastasis.

Tetraspanin 2 (Tspan2) is one of the less well-characterised members of the tetraspanin superfamily, and its precise function in different human tissue types remains to be explored. Initial studies have highlighted its possible association in neuroinflammation and carcinogenesis. In the central nervous system, Tspan2 may contribute to the early stages of the oligodendrocyte differentiation into myelin-forming glia. Furthermore, in human lung cancer, Tspan2 could be involved in the progression of the tumour metastasis by modulating cancer cell motility and invasion functions. In this review, we discuss the available evidence for the potential role of Tspan2 and introduce possible strategies for disease targeting.

Peptides from Tetraspanin CD9 Are Potent Inhibitors of Staphylococcus Aureus Adherence to Keratinocytes.

Staphylococcus aureus is one of the primary causative agents of skin and wound infections. As bacterial adherence is essential for infection, blocking this step can reduce invasion of host tissues by pathogens. An anti-adhesion therapy, based on a host membrane protein family, the tetraspanins, has been developed that can inhibit the adhesion of S. aureus to human cells. Synthetic peptides derived from a keratinocyte-expressed tetraspanin, CD9, were tested for anti-adhesive properties and at low nanomolar concentrations were shown to inhibit bacterial adhesion to cultured keratinocytes and to be effective in a tissue engineered model of human skin infection. These potential therapeutics had no effect on keratinocyte viability, migration or proliferation, indicating that they could be a valuable addition to current treatments for skin infection.

Distinct regions of the large extracellular domain of tetraspanin CD9 are involved in the control of human multinucleated giant cell formation.

Multinucleated giant cells, formed by the fusion of monocytes/macrophages, are features of chronic granulomatous inflammation associated with infections or the persistent presence of foreign material. The tetraspanins CD9 and CD81 regulate multinucleated giant cell formation: soluble recombinant proteins corresponding to the large extracellular domain (EC2) of human but not mouse CD9 can inhibit multinucleated giant cell formation, whereas human CD81 EC2 can antagonise this effect. Tetraspanin EC2 are all likely to have a conserved three helix sub-domain and a much less well-conserved or hypervariable sub-domain formed by short helices and interconnecting loops stabilised by two or more disulfide bridges. Using CD9/CD81 EC2 chimeras and point mutants we have mapped the specific regions of the CD9 EC2 involved in multinucleated giant cell formation. These were primarily located in two helices, one in each sub-domain. The cysteine residues involved in the formation of the disulfide bridges in CD9 EC2 were all essential for inhibitory activity but a conserved glycine residue in the tetraspanin-defining 'CCG' motif was not. A tyrosine residue in one of the active regions that is not conserved between human and mouse CD9 EC2, predicted to be solvent-exposed, was found to be only peripherally involved in this activity. We have defined two spatially-distinct sites on the CD9 EC2 that are required for inhibitory activity. Agents that target these sites could have therapeutic applications in diseases in which multinucleated giant cells play a pathogenic role.

Tetraspanins: gateways for infection.

The tetraspanins constitute a conserved superfamily of four-span membrane proteins that are widely distributed in multi-cellular organisms. A characteristic property of tetraspanins is their ability to form lateral associations with one another and with other membrane proteins, giving rise to tetraspanin enriched microdomains (TEM) that are involved in the molecular organisation of many membrane-associated functions such as adhesion, fusion and trafficking. Increasing evidence suggests that intracellular pathogens, especially viruses, have "hijacked" tetraspanins as a means of entering, traversing and exiting cells during the course of infection. This article reviews current evidence for the role of tetraspanins in the uptake, trafficking and spread of viruses as well as intracellular bacteria, fungi and parasites. Finally, the prospects of targeting tetraspanins for therapeutic intervention in infections caused by such pathogens are discussed.

A Burkholderia pseudomallei toxin inhibits helicase activity of translation factor eIF4A.

The structure of BPSL1549, a protein of unknown function from Burkholderia pseudomallei, reveals a similarity to Escherichia coli cytotoxic necrotizing factor 1. We found that BPSL1549 acted as a potent cytotoxin against eukaryotic cells and was lethal when administered to mice. Expression levels of bpsl1549 correlate with conditions expected to promote or suppress pathogenicity. BPSL1549 promotes deamidation of glutamine-339 of the translation initiation factor eIF4A, abolishing its helicase activity and inhibiting translation. We propose to name BPSL1549 Burkholderia lethal factor 1.

Regulation of zebrafish hatching by tetraspanin cd63.

Tetraspanins cause the clustering of membrane proteins into a level of organisation essential for cellular function. Given the importance and complicated nature of this mechanism, we attempted a novel approach to identify the function of a single component in a biologically relevant context. A morpholino knockdown strategy was used to investigate the role of cd63, a membrane protein associated with intracellular transport and a melanoma marker, in embryonic zebrafish. By using three separate morpholinos targeting cd63, we were able to identify a specific phenotype. Strikingly, morphant fish failed to hatch due to the lack of secreted proteolytic enzymes required for chorion-softening. The morphology of the hatching gland at both the cellular and intracellular levels was disorganised, suggesting a role for cd63 in the functioning of this organ. This work identifies a specific role for cd63 in the zebrafish embryo and provides evidence for the suitability of zebrafish as a model system for the investigation of tetraspanin enriched microdomains.

Cooperative role for tetraspanins in adhesin-mediated attachment of bacterial species to human epithelial cells.

The tetraspanins are a superfamily of transmembrane proteins with diverse functions and can form extended microdomains within the plasma membrane in conjunction with partner proteins, which probably includes receptors for bacterial adhesins. Neisseria meningitidis, the causative agent of meningococcal disease, attaches to host nasopharyngeal epithelial cells via type IV pili and opacity (Opa) proteins. We examined the role of tetraspanin function in Neisseria meningitidis adherence to epithelial cells. Tetraspanins CD9, CD63, and CD151 were expressed by HEC-1-B and DETROIT 562 cells. Coincubation of cells with antibodies against all three tetraspanin molecules used individually or in combination, with recombinant tetraspanin extracellular domains (EC2), or with small interfering RNAs (siRNAs) significantly reduced adherence of Neisseria meningitidis. In contrast, recombinant CD81, a different tetraspanin, had no effect on meningococcal adherence. Antitetraspanin antibodies reduced the adherence to epithelial cells of Neisseria meningitidis strain derivatives expressing Opa and pili significantly more than isogenic strains lacking these determinants. Adherence to epithelial cells of strains of Staphylococcus aureus, Neisseria lactamica, Escherichia coli, and Streptococcus pneumoniae was also reduced by pretreatment of cells with tetraspanin antibodies and recombinant proteins. These data suggest that tetraspanins are required for optimal function of epithelial adhesion platforms containing specific receptors for Neisseria meningitidis and potentially for multiple species of bacteria.