Bacterial invasion via lipid rafts

Accumulating reports document the use by pathogens of cholesterol‐enriched lipid microdomains, often called lipid rafts, as cell surface platforms to interact, bind and possibly enter into host cells. The challenge is now to understand what could be the functional role of these domains during pathogen invasion. Are they hijacked as general clustering devices for cellular binding sites and/or do they have other roles? In particular, is their cell signalling capacity activated and used by pathogens? In reverse, could lipid rafts activate bacterial mechanisms required for invasion? These issues will be discussed after an introduction on the current view on lipid rafts.     

Human immunodeficiency virus type 1 Nef activates p21-activated kinase via recruitment into lipid rafts

The Nef protein of human immunodeficiency virus type 1 is an important factor in AIDS pathogenesis. In addition to downregulating CD4 and major histocompatibility complex class I molecules from the cell surface, as well as increasing virion infectivity, Nef triggers activation of the T-cell receptor (TCR) cascade to facilitate virus spread. Signaling pathways that are induced by Nef have been identified; however, it is unclear how and in which subcellular compartment Nef triggers signaling. Nef recruits a multiprotein complex to activate the cellular Pak kinase that mediates downstream effector functions. Since a subpopulation of Nef is present in detergent-insoluble microdomains (lipid rafts) from where physiological TCR signaling is initiated, we tested whether lipid rafts are instrumental for Nef-mediated Pak activation. In flotation analysis, Nef-associated Pak activity exclusively fractionated with lipid rafts. Activation of Pak in the presence of Nef coincided with lipid raft recruitment of the kinase, which was otherwise excluded from detergent-insoluble microdomains. Experimental solubilization of lipid rafts interfered with the association of Pak activity with Nef. To analyze the importance of the raft localization for Nef function more rigorously, we generated a palmitoylated Nef (PalmNef). PalmNef was highly enriched in lipid rafts and associated with significantly higher levels of Pak activity than Nef. Notably, activation of Pak by its physiological activators, Cdc42 and Rac, also occurred in lipid rafts and required raft integrity. Together, these data suggest that Nef induces signal transduction via the recruitment of a signaling machinery including Pak into lipid rafts, thereby mimicking a physiological cellular mechanism to initiate the TCR cascade.     

Matrix fibronectin increases HIV stability and infectivity

HIV particles are detected extracellularly in lymphoid tissues, a major reservoir of the virus. We previously reported that a polymerized form of fibronectin (FN), superfibronectin (sFN), as well as a fragment of FN, III1-C, enhanced infection of primary CD4(+) T cells by HIV-1IIIB. We now show that sFN enhances infection of primary CD4(+) T cells by both R5 and X4 strains of HIV-1. Using HIV pseudotyped with different envelope glycoproteins (gp120) and HOS cells transfected with various chemokine receptors alone or in combination with the CD4 molecule, we show that sFN-mediated enhancement requires the CD4 receptor and does not alter the specificity of gp120 for different chemokine receptors. Because the III1-C fragment also resulted in enhancement, we asked whether proteolysis of FN generated fragments capable of enhancing HIV infection. We found that progressive proteolysis of FN by chymotrypsin correlates with an enhancement of HIV infection in both primary CD4(+) T cells and the IG5 reporter cell line. Furthermore, incubation of HIV with sFN significantly prolonged infectivity at 37 degrees C compared with dimeric FN or BSA. In conclusion, these results indicate that polymerized (matrix) or degraded (inflammation-associated), but not dimeric (plasma), FN are capable of enhancing infection by HIV-1, independent of the coreceptor specificity of the strains. Moreover, virions bound to matrix FN maintain infectivity for longer periods of time than do virions in suspension. This study suggests that matrix proteins and their conformational status may play a role in the pathogenesis of HIV.     

HIV Nef increases T cell ERK MAP kinase activity.

The human immunodeficiency regulatory protein Nef enhances viral replication and is central to viral pathogenesis. Although Nef has displayed a capacity to associate with a diverse assortment of cellular molecules and to increase T cell activity, the biochemical activity of Nef in T cells remains poorly defined. In this report we examine the bioactivity of Nef in primary CD4 T cells and, in particular, focus on the biochemical pathways known to be central to T cell activity. The extracellular signal-regulated kinase (ERK) mitogen-activated protein (MAP) kinase pathway was dramatically affected by Nef expression with increases in ERK, MEK, and Elk induction. The capacity of Nef to increase the MAP kinase pathway activity was dependent on T cell receptor stimulation. By increasing ERK MAP kinase activity, Nef is functionally associated with a kinase known to affect T cell activity, viral replication, and viral infectivity.     

Functional roles of glycosphingolipids in signal transduction via lipid rafts

The formation of glycosphingolipid (GSL)-cholesterol microdomains in cell membranes has been proposed to function as platforms for the attachment of lipid-modified proteins, such as glycosylphosphatidylinositol (GPI)-anchored proteins and src-family tyrosine kinases. The microdomains are postulated to be involved in GPI-anchored protein signaling via src-family kinase. Here, the functional roles of GSLs in signal transduction mediated by the microdomains are discussed. Antibodies against GSLs co-precipitate GPI-anchored proteins, src-family kinases and several components of the microdomains. Antibody-mediated crosslinking of GSLs, as well as that of GPI-anchored proteins, induces a rapid activation of src-family kinases and a transient increase in the tyrosine phosphorylation of several substrates. Enzymatic degradation of GSLs reduces the activation of src-family kinase and tyrosine phosphorylation by antibody-mediated crosslinking of GPI-anchored protein. Furthermore, GSLs can also modulate signal transduction of immunoreceptors and growth factor receptors in the microdomains. Thus, GSLs have important roles in signal transduction mediated by the microdomains.     

Functional roles of glycosphingolipids in signal transduction via lipid rafts

The formation of glycosphingolipid (GSL)-cholesterol microdomains in cell membranes has been proposed to function as platforms for the attachment of lipid-modified proteins, such as glycosylphosphatidylinositol (GPI)-anchored proteins and src-family tyrosine kinases. The microdomains are postulated to be involved in GPI-anchored protein signaling via src-family kinase. Here, the functional roles of GSLs in signal transduction mediated by the microdomains are discussed. Antibodies against GSLs co-precipitate GPI-anchored proteins, src-family kinases and several components of the microdomains. Antibody-mediated crosslinking of GSLs, as well as that of GPI-anchored proteins, induces a rapid activation of src-family kinases and a transient increase in the tyrosine phosphorylation of several substrates. Enzymatic degradation of GSLs reduces the activation of src-family kinase and tyrosine phosphorylation by antibody-mediated crosslinking of GPI-anchored protein. Furthermore, GSLs can also modulate signal transduction of immunoreceptors and growth factor receptors in the microdomains. Thus, GSLs have important roles in signal transduction mediated by the microdomains.     

Depletion of cellular cholesterol and lipid rafts increases shedding of CD30

CD30, a lymphoid activation marker, is shed into the cell environment after endoproteolytic cleavage of its ectodomain. Soluble (s)CD30 is able to suppress the Th1-type immune response. Because high serum levels of sCD30 and cholesterol-lowering drugs seem to be beneficial in some Th1-type autoimmune diseases, we focused on a link between CD30 shedding and the amount of cellular cholesterol. Cholesterol depletion of human Hodgkin lymphoma- and non-Hodgkin lymphoma-derived cell lines by methyl-beta-cyclodextrin led to a down-regulation of membrane-bound CD30 and increased release of sCD30. Additionally, the cholesterol-interfering drugs lovastatin, cholesterol oxidase, and filipin increased CD30 shedding. Both the down-regulation of membrane-anchored CD30 and the release of sCD30 were dependent on metalloproteinases. Using specific inhibitors, we detected TNF-alpha converting enzyme (TACE) as the leading enzyme responsible for cholesterol-dependent CD30 shedding. A Triton X-100-based method for lipid raft isolation revealed that CD30 was partially present in lipid rafts, whereas TACE was localized in the nonraft fractions. Disintegration of lipid rafts by cholesterol depletion might therefore lead to dynamic interactions of CD30 with TACE, resulting in enhanced shedding of CD30. Our results suggest a possible role of cholesterol-dependent shedding of CD30 in the pathogenesis of immune diseases.     

Human immunodeficiency virus type 1 Nef associates with lipid rafts to downmodulate cell surface CD4 and class I major histocompatibility complex expression and to increase viral infectivity

Lipid rafts are membrane microdomains that are functionally distinct from other membrane regions. We have shown that 10% of human immunodeficiency virus type 1 (HIV-1) Nef expressed in SupT1 cells is present in lipid rafts and that this represents virtually all of the membrane-associated Nef. To determine whether raft targeting, rather than simply membrane localization, has functional significance, we created a Nef fusion protein (LAT-Nef) containing the N-terminal 35 amino acids from LAT, a protein that is exclusively localized to rafts. Greater than 90% of the LAT-Nef protein was found in the raft fraction. In contrast, a mutated form, lacking two cysteine palmitoylation sites, showed less than 5% raft localization. Both proteins were equally expressed and targeted nearly exclusively to membranes. The LAT-Nef protein was more efficient than its nonraft mutant counterpart at downmodulating both cell surface CD4 and class I major histocompatibility complex (MHC) expression, as well as in enhancing first-round infectivity and being incorporated into virus particles. This demonstrates that targeting of Nef to lipid rafts is mechanistically important for all of these functions. Compared to wild-type Nef, LAT-Nef downmodulated class I MHC nearly as effectively as the wild-type Nef protein, but was only about 60% as effective for CD4 downmodulation and 30% as effective for infectivity enhancement. Since the LAT-Nef protein was found entirely in rafts while the wild-type Nef protein was distributed 10% in rafts and 90% in the soluble fraction, our results suggest that class I MHC downmodulation by Nef may be performed exclusively by raft-bound Nef. In contrast, CD4 downmodulation and infectivity enhancement may require a non-membrane-bound Nef component as well as the membrane-bound form.     

Imaging lipid rafts

Lipid rafts are plasma membrane microdomains enriched in sphingolipids and cholesterol. These domains have been suggested to serve as platforms for various cellular events, such as signaling and membrane trafficking. However, little is known about the distribution and dynamics of lipids in these microdomains. Here we report investigations carried out using recently developed probes for the lipid components of lipid rafts: lysenin, a sphingomyelin-binding protein obtained from the coelomic fluid of the earthworm Eisenia foetida; and the fluorescein ester of poly(ethyleneglycol) cholesteryl ether (fPEG-Chol), which partitions into cholesterol-rich membranes. Lysenin reveals that the organization of sphingomyelin differs between different cell types and even between different membrane domains within the same cell. When added to live cells, fPEG-Chol is distributed exclusively on the outer leaflet of the plasma membrane and is clustered dynamically upon activation of receptor signaling. The surface-bound fPEG-Chol is slowly internalized via a clathrin-independent pathway into endosomes with lipid raft markers.     

The effects of HIV-1 Nef on CD4 surface expression and viral infectivity in lymphoid cells are independent of rafts

The HIV-1 Nef protein is a critical virulence factor that exerts multiple effects during viral replication. Nef modulates surface expression of various cellular proteins including CD4 and MHC-I, enhances viral infectivity, and affects signal transduction pathways. Nef has been shown to partially associate with rafts, where it can prime T cells for activation. The contribution of rafts during Nef-induced CD4 down-regulation and enhancement of viral replication remains poorly understood. We show here that Nef does not modify the palmitoylation state of CD4 or its partition within rafts. Moreover, CD4 mutants lacking palmitoylation or unable to associate with rafts are efficiently down-regulated by Nef. In HIV-infected cells, viral assembly and budding occurs from rafts, and Nef has been suggested to increase this process. However, using T cells acutely infected with wild-type or nef-deleted HIV, we did not observe any impact of Nef on raft segregation of viral structural proteins. We have also designed a palmitoylated mutant of Nef (NefG3C), which significantly accumulates in rafts. Interestingly, the efficiency of NefG3C to down-regulate CD4 and MHC-I, and to promote viral replication was not increased when compared with the wild-type protein. Altogether, these results strongly suggest that rafts are not a key element involved in the effects of Nef on trafficking of cellular proteins and on viral replication.     

Caveolin-1 reduces HIV-1 infectivity by restoration of HIV Nef mediated impairment of cholesterol efflux by apoA-I

Background

Transmitted HIV-1 clade B or C R5 viruses have been reported to infect macrophages inefficiently, while other studies have described R5 viruses in late disease with either an enhanced macrophage-tropism or carrying envelopes with an increased positive charge and fitness. In contrast, our previous data suggested that viruses carrying non-macrophage-tropic R5 envelopes were still predominant in immune tissue of AIDS patients. To further investigate the tropism and charge of HIV-1 viruses in late disease, we evaluated the properties of HIV-1 envelopes amplified from immune and brain tissues of AIDS patients with neurological complications.

Results

Almost all envelopes amplified were R5. There was clear compartmentalization of envelope sequences for four of the five subjects. However, strong compartmentalization of macrophage-tropism in brain was observed even when brain and immune tissue envelope sequences were not segregated. R5 envelopes from immune tissue of four subjects carried a higher positive charge compared to brain envelopes. We also confirm a significant correlation between macrophage tropism and sensitivity to soluble CD4, a weak association with sensitivity to the CD4 binding site antibody, b12, but no clear relationship with maraviroc sensitivity.

Conclusions

Our study shows that non-macrophage-tropic R5 envelopes carrying gp120s with an increased positive charge were predominant in immune tissue in late disease. However, highly macrophage-tropic variants with lower charged gp120s were nearly universal in the brain. These results are consistent with HIV-1 R5 envelopes evolving gp120s with an increased positive charge in immune tissue or sites outside the brain that likely reflect an adaptation for increased replication or fitness for CD4+ T-cells. Our data are consistent with the presence of powerful pressures in brain and in immune tissues selecting for R5 envelopes with very different properties; high macrophage-tropism, sCD4 sensitivity and low positive charge in brain and non-macrophage-tropism, sCD4 resistance and high positive charge in immune tissue.     

uPA binding increases UPAR localization to lipid rafts and modifies the receptor microdomain composition

UPAR is a GPI anchored protein, which is found in both lipid rafts and in more fluid regions of the plasma membrane. We have studied the role of the ligand uPA on uPAR localization and on the composition of the lipid membrane microdomains. We have analyzed the glycosphingolipid environment of uPAR in detergent resistant membrane (DRM) fractions prepared by cell lysis with 1% Triton X-100 and fractionated by sucrose gradient centrifugation obtained from HEK293-uPAR cells. The uPAR specific lipid membrane microdomain has been separated from the total DRM fraction by immunoprecipitation with an anti-uPAR specific antibody under conditions that preserve membrane integrity. We have also tested uPA-induced ERK phosphorylation in the presence of methyl-β-cyclodextrin, which is known to disrupt lipid rafts by sequestering cholesterol from such domains. Our results show that uPAR is partially associated with DRM and this association is increased by ligands, is independent of the catalytic activity of uPA, and is required for intracellular signalling. In the absence of ligands, uPAR experiences a lipid environment very similar to that of total DRM, enriched in sphingomyelin and glycosphingolipids. However, after treatment of cells with uPA or ATF the lipid environment is strongly impoverished of neutral glycosphingolipids.     

uPA binding increases UPAR localization to lipid rafts and modifies the receptor microdomain composition

UPAR is a GPI anchored protein, which is found in both lipid rafts and in more fluid regions of the plasma membrane. We have studied the role of the ligand uPA on uPAR localization and on the composition of the lipid membrane microdomains. We have analyzed the glycosphingolipid environment of uPAR in detergent resistant membrane (DRM) fractions prepared by cell lysis with 1% Triton X-100 and fractionated by sucrose gradient centrifugation obtained from HEK293-uPAR cells. The uPAR specific lipid membrane microdomain has been separated from the total DRM fraction by immunoprecipitation with an anti-uPAR specific antibody under conditions that preserve membrane integrity. We have also tested uPA-induced ERK phosphorylation in the presence of methyl-beta-cyclodextrin, which is known to disrupt lipid rafts by sequestering cholesterol from such domains. Our results show that uPAR is partially associated with DRM and this association is increased by ligands, is independent of the catalytic activity of uPA, and is required for intracellular signalling. In the absence of ligands, uPAR experiences a lipid environment very similar to that of total DRM, enriched in sphingomyelin and glycosphingolipids. However, after treatment of cells with uPA or ATF the lipid environment is strongly impoverished of neutral glycosphingolipids.     

Bacterial subversion of lipid rafts

Bacteria rely on numerous basic cellular functions of their target cells to reach successful infection. The recent discovery that the plasma membrane contains specialized microdomains, called lipid rafts, with many specific functions but in particular with the ability to concentrate signaling molecules, has therefore attracted the attention of cellular microbiologists. Since then an increasing number of bacteria and their products have been shown to interact with lipid rafts to promote infection or intoxication. Here we review why certain bacteria and/or their products are attracted toward these lipid microdomains.     

Aggregation of lipid rafts accompanies signaling via the T cell antigen receptor.

The role of lipid rafts in T cell antigen receptor (TCR) signaling was investigated using fluorescence microscopy. Lipid rafts labeled with cholera toxin B subunit (CT-B) and cross-linked into patches displayed characteristics of rafts isolated biochemically, including detergent resistance and colocalization with raft-associated proteins. LCK, LAT, and the TCR all colocalized with lipid patches, although TCR association was sensitive to nonionic detergent. Aggregation of the TCR by anti-CD3 mAb cross-linking also caused coaggregation of raft-associated proteins. However, the protein tyrosine phosphatase CD45 did not colocalize to either CT-B or CD3 patches. Cross-linking of either CD3 or CT-B strongly induced tyrosine phosphorylation and recruitment of a ZAP-70(SH2)(2)-green fluorescent protein (GFP) fusion protein to the lipid patches. Also, CT-B patching induced signaling events analagous to TCR stimulation, with the same dependence on expression of key TCR signaling molecules. Targeting of LCK to rafts was necessary for these events, as a nonraft- associated transmembrane LCK chimera, which did not colocalize with TCR patches, could not reconstitute CT-B-induced signaling. Thus, our results indicate a mechanism whereby TCR engagement promotes aggregation of lipid rafts, which facilitates colocalization of LCK, LAT, and the TCR whilst excluding CD45, thereby triggering protein tyrosine phosphorylation.     

Uptake of granulysin via lipid rafts leads to lysis of intracellular Listeria innocua

The bacteriolytic activity of CTL is mediated by granulysin, which has been reported to kill intracellular Mycobacterium tuberculosis in dendritic cells (DC) with high efficiency. Despite that crucial effector function, the killing mechanism and uptake of granulysin into target cells have not been well investigated. To this end we analyzed granulysin binding, uptake, and the subsequent lysis of intracellular Listeria innocua in human DC. Recombinant granulysin was found to be actively taken up by DC into early endosomal Ag 1-labeled endosomes, as detected by immunofluorescence. Further transfer to L. innocua-containing phagosomes was indicated by colocalization of bacterial DNA with granulysin. After uptake of granulysin by DC, lysis of L. innocua was found in a dose-dependent manner. Uptake as well as lysis of Listeria were inhibited after blocking endocytosis by lowering the temperature and by cholesterol depletion of DC. Colocalization of granulysin with cholera toxin during uptake showed binding to and internalization via lipid rafts. In contrast to cholera toxin, which was targeted to the perinuclear compartment, granulysin was found exclusively in endosomal-phagosomal vesicles. Lipid raft microdomains, enriched in the immunological synapse, may thus enhance uptake and transfer of granulysin into bacterial infected host cells.     

Nef associates with p21-activated kinase 2 in a p21-GTPase-dependent dynamic activation complex within lipid rafts

We have previously reported that Nef specifically interacts with a small but highly active subpopulation of p21-activated kinase 2 (PAK2). Here we show that this is due to a transient association of Nef with a PAK2 activation complex within a detergent-insoluble membrane compartment containing the lipid raft marker GM1. The low abundance of this Nef-associated kinase (NAK) complex was found to be due to an autoregulatory mechanism. Although activation of PAK2 was required for assembly of the NAK complex, catalytic activity of PAK2 also promoted dissociation of this complex. Testing different constitutively active PAK2 mutants indicated that the conformation associated with p21-mediated activation rather than kinase activity per se was required for PAK2 to become NAK. Although association with PAK2 is one of the most conserved properties of Nef, we found that the ability to stimulate PAK2 activity differed markedly among divergent Nef alleles, suggesting that PAK2 association and activation are distinct functions of Nef. However, mutations introduced into the p21-binding domain of PAK2 revealed that p21-GTPases are involved in both of these Nef functions and, in addition to promoting PAK2 activation, also help to physically stabilize the NAK complex.     

Membrane organization and lipid rafts

Cell membranes are composed of a lipid bilayer, containing proteins that span the bilayer and/or interact with the lipids on either side of the two leaflets. Although recent advances in lipid analytics show that membranes in eukaryotic cells contain hundreds of different lipid species, the function of this lipid diversity remains enigmatic. The basic structure of cell membranes is the lipid bilayer, composed of two apposing leaflets, forming a two-dimensional liquid with fascinating properties designed to perform the functions cells require. To coordinate these functions, the bilayer has evolved the propensity to segregate its constituents laterally. This capability is based on dynamic liquid-liquid immiscibility and underlies the raft concept of membrane subcompartmentalization. This principle combines the potential for sphingolipid-cholesterol self-assembly with protein specificity to focus and regulate membrane bioactivity. Here we will review the emerging principles of membrane architecture with special emphasis on lipid organization and domain formation.